Evolutionary Relationship of Disease Resistance Genes in Soybean and Arabidopsis Specific for the Pseudomonas syringae Effectors AvrB and AvrRpm1

In Arabidopsis (Arabidopsis thaliana), the Pseudomonas syringae effector proteins AvrB and AvrRpm1 are both detected by the RESISTANCE TO PSEUDOMONAS MACULICOLA1 (RPM1) disease resistance (R) protein. By contrast, soybean (Glycine max) can distinguish between these effectors, with AvrB and AvrRpm1 being detected by the Resistance to Pseudomonas glycinea 1b (Rpg1b) and Rpg1r R proteins, respectively. We have been using these genes to investigate the evolution of R gene specificity and have previously identified RPM1 and Rpg1b. Here, we report the cloning of Rpg1r, which, like RPM1 and Rpg1b, encodes a coiled-coil (CC)-nucleotide-binding (NB)-leucine-rich repeat (LRR) protein. As previously found for Rpg1b, we determined that Rpg1r is not orthologous with RPM1, indicating that the ability to detect both AvrB and AvrRpm1 evolved independently in soybean and Arabidopsis. The tightly linked soybean Rpg1b and Rpg1r genes share a close evolutionary relationship, with Rpg1b containing a recombination event that combined a NB domain closely related to Rpg1r with CC and LRR domains from a more distantly related CC-NB-LRR gene. Using structural modeling, we mapped polymorphisms between Rpg1b and Rpg1r onto the predicted tertiary structure of Rpg1b, which revealed highly polymorphic surfaces within both the CC and LRR domains. Assessment of chimeras between Rpg1b and Rpg1r using a transient expression system revealed that AvrB versus AvrRpm1 specificity is determined by the C-terminal portion of the LRR domain. The P. syringae effector AvrRpt2, which targets RPM1 INTERACTOR4 (RIN4) proteins in both Arabidopsis and soybean, partially blocked recognition of both AvrB and AvrRpm1 in soybean, suggesting that both Rpg1b and Rpg1r may detect these effectors via modification of a RIN4 homolog.Effector triggered immunity in plants involves highly specific recognition events in which plant resistance (R) proteins detect pathogen effector proteins directly or, alternatively, the modifications that they induce on host proteins (Bonardi et al., 2012). The largest group of R proteins belongs to the nucleotide-binding (NB)-leucine-rich repeat (LRR) family (McHale et al., 2006). The NB-LRR family can be further subdivided based on N-terminal domains into the Toll-Interleukin and R protein (TIR) class and non-TIR-NB-LRR class (McHale et al., 2006). The latter most often contain a coiled-coil (CC) domain at the N terminus. The contributions of the TIR, CC, and LRR domains to R protein specificity, and how new specificities evolve, remain important questions.There are relatively few NB-LRR R proteins characterized to date that are thought to detect pathogen effectors directly; these include Pi-ta from rice (Oryza sativa), L and M variants from flax (Linum usitatissimum), and RESISTANCE TO RALSTONIA SOLANACEARUM1 and RESISTANCE TO PERONOSPORA PARASITICA1 (RPP1) from Arabidopsis (Arabidopsis thaliana; Jia et al., 2000; Deslandes et al., 2003; Dodds et al., 2006; Ueda et al., 2006; Catanzariti et al., 2010; Krasileva et al., 2010). In at least some of these examples, the R genes are found in clusters of NB-LRR paralogs in which multiple recognition specificities are represented (Ellis et al., 1995; Botella et al., 1998) or belong to allelic series (Ellis et al., 1995), arrangements that may promote evolution of recognition specificity via recombination between alleles and paralogs. Interestingly, sequence comparisons and domain swaps involving alleles at the L locus implicate both the LRR and TIR regions as determinants of recognition specificity (Ellis et al., 1999; Luck et al., 2000). Subsequently, domain swaps involving paralogs clustered at the barley (Hordeum vulgare) MILDEW A (MLA) and potato (Solanum tuberosum) Resistance to Potato Virus X (Rx)/Globodera pallida (Gpa) loci have provided additional support for the LRR domain playing a key role in conferring recognition specificity (Ellis et al., 1999; Luck et al., 2000; Shen et al., 2003; Rairdan and Moffett, 2006).Several R proteins are known to detect the presence of pathogen effectors indirectly by monitoring the activity of pathogen effectors within the plant cell. For example, the Arabidopsis RESISTANCE TO PSEUDOMONAS MACULICOLA1 (RPM1) and RESISTANCE TO PSEUDOMONAS SYRINGAE2 (RPS2) R proteins detect modification of the effector target RPM1 INTERACTOR4 (RIN4), while the Arabidopsis RPS5 protein detects modification of the effector target AvrPphB SUSCEPTIBLE1 (Mackey et al., 2002, 2003; Axtell and Staskawicz, 2003; Shao et al., 2003). At least for the well-studied examples in Arabidopsis, R proteins that employ indirect recognition mechanisms are encoded by NB-LRR genes that are not members of large clusters, or allelic series, with variants encoding distinct recognition specificities. Correlated with this genomic structure, such loci are typically relatively stable, with RPM1 and RPS5 existing as presence/absence polymorphisms that have been maintained over long evolutionary periods (Stahl et al., 1999; Tian et al., 2002). Both functional and nonfunctional alleles of RPS2 have been isolated, but only a single recognition specificity has been detected at this locus, despite sequence polymorphisms between alleles (Caicedo et al., 1999).Most likely, specificity for this class of R proteins is determined by a combination of the ability to associate with the host protein targeted by the effector and the ability to detect effector-induced modification of this target. Consistent with this hypothesis, it has been shown that the CC domains from at least some R proteins interact with the host proteins they are monitoring, even in the absence of pathogen effectors, in a prerecognition complex (Mackey et al., 2002; Ade et al., 2007). Hence, evolution of recognition specificity in R proteins that employ indirect recognition mechanisms may involve evolution of both the N-terminal CC and LRR domains.To better understand the evolution and function of R proteins that detect pathogen effectors indirectly, we have been studying two soybean (Glycine max) R genes, with known recognition specificities, that are members of a complex NB-LRR cluster. The R genes involved, Resistance to Pseudomonas glycinea 1b (Rpg1b) and Rpg1r, mediate detection of the Pseudomonas syringae effector proteins AvrB and AvrRpm1, respectively (Staskawicz et al., 1984; Ashfield et al., 1995). We have previously cloned Rpg1b, which is a CC-NB-LRR (CNL) gene that maps to a cluster of R genes effective against a diverse range of pathogens (Ashfield et al., 1998, 2004). Rpg1r is present in the same cluster and maps 0.56 centiMorgans from Rpg1b (Ashfield et al., 1995); however, the evolutionary relationship shared by the two R genes is not known. The cluster is associated with numerous NB-LRR genes, of both the CC and TIR subgroups, spread over more than a megabase of soybean chromosome 13 (Peñuela et al., 2002; Hayes et al., 2004; Innes et al., 2008; Ashfield et al., 2012; Wen et al., 2013). The NB-LRR family in this region is evolving rapidly, with duplications/deletions of paralogs, recombination, and positive selection all playing a role (Ashfield et al., 2012).While soybean can distinguish between AvrB and AvrRpm1, both effectors are detected by a single R protein, RPM1, in Arabidopsis (Bisgrove et al., 1994; Grant et al., 1995). It is known that RPM1 recognizes the effector proteins indirectly by detecting effector-dependent phosphorylation of a second Arabidopsis protein, RIN4 (Mackey et al., 2002; Chung et al., 2011; Liu et al., 2011). The available evidence suggests that a related strategy is employed by soybean, at least for the Rpg1b protein, despite the AvrB recognition specificity having evolved independently in these plant species (Ashfield et al., 2004; Selote and Kachroo, 2010; Selote et al., 2013). Soybean contains four RIN4 homologs (Chen et al., 2010), three of which interact physically with Rpg1b, with two required for full resistance conferred by this R gene (Selote and Kachroo, 2010; Selote et al., 2013). It is not known whether RIN4 homologs are required for Rpg1r function.Here, we report the map-based cloning of the soybean Rpg1r gene. Comparison of the Rpg1r protein to Rpg1b, combined with structural modeling, revealed highly polymorphic surfaces in the CC and LRR domains. Transient expression of chimeric Rpg1 proteins demonstrated that specificity for AvrB versus AvrRpm1 is determined by the C-terminal LRR region. Finally, we provide evidence that Rpg1r, like Rpg1b, detects its corresponding pathogen effector indirectly, most likely by monitoring a RIN4 homolog, indicating convergent evolution of recognition mechanisms in separate plant families.     

Natural Variation in Small Molecule–Induced TIR-NB-LRR Signaling Induces Root Growth Arrest via EDS1- and PAD4-Complexed R Protein VICTR in Arabidopsis

In a chemical genetics screen we identified the small-molecule [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione (DFPM) that triggers rapid inhibition of early abscisic acid signal transduction via PHYTOALEXIN DEFICIENT4 (PAD4)- and ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)-dependent immune signaling mechanisms. However, mechanisms upstream of EDS1 and PAD4 in DFPM-mediated signaling remain unknown. Here, we report that DFPM generates an Arabidopsis thaliana accession-specific root growth arrest in Columbia-0 (Col-0) plants. The genetic locus responsible for this natural variant, VICTR (VARIATION IN COMPOUND TRIGGERED ROOT growth response), encodes a TIR-NB-LRR (for Toll-Interleukin1 Receptor–nucleotide binding–Leucine-rich repeat) protein. Analyses of T-DNA insertion victr alleles showed that VICTR is necessary for DFPM-induced root growth arrest and inhibition of abscisic acid–induced stomatal closing. Transgenic expression of the Col-0 VICTR allele in DFPM-insensitive Arabidopsis accessions recapitulated the DFPM-induced root growth arrest. EDS1 and PAD4, both central regulators of basal resistance and effector-triggered immunity, as well as HSP90 chaperones and their cochaperones RAR1 and SGT1B, are required for the DFPM-induced root growth arrest. Salicylic acid and jasmonic acid signaling pathway components are dispensable. We further demonstrate that VICTR associates with EDS1 and PAD4 in a nuclear protein complex. These findings show a previously unexplored association between a TIR-NB-LRR protein and PAD4 and identify functions of plant immune signaling components in the regulation of root meristematic zone-targeted growth arrest.     

Long-Term Evolution of Nucleotide-Binding Site-Leucine-Rich Repeat Genes: Understanding Gained from and beyond the Legume Family

Proper utilization of plant disease resistance genes requires a good understanding of their short- and long-term evolution. Here we present a comprehensive study of the long-term evolutionary history of nucleotide-binding site (NBS)-leucine-rich repeat (LRR) genes within and beyond the legume family. The small group of NBS-LRR genes with an amino-terminal RESISTANCE TO POWDERY MILDEW8 (RPW8)-like domain (referred to as RNL) was first revealed as a basal clade sister to both coiled-coil-NBS-LRR (CNL) and Toll/Interleukin1 receptor-NBS-LRR (TNL) clades. Using Arabidopsis (Arabidopsis thaliana) as an outgroup, this study explicitly recovered 31 ancestral NBS lineages (two RNL, 21 CNL, and eight TNL) that had existed in the rosid common ancestor and 119 ancestral lineages (nine RNL, 55 CNL, and 55 TNL) that had diverged in the legume common ancestor. It was shown that, during their evolution in the past 54 million years, approximately 94% (112 of 119) of the ancestral legume NBS lineages experienced deletions or significant expansions, while seven original lineages were maintained in a conservative manner. The NBS gene duplication pattern was further examined. The local tandem duplications dominated NBS gene gains in the total number of genes (more than 75%), which was not surprising. However, it was interesting from our study that ectopic duplications had created many novel NBS gene loci in individual legume genomes, which occurred at a significant frequency of 8% to 20% in different legume lineages. Finally, by surveying the legume microRNAs that can potentially regulate NBS genes, we found that the microRNA-NBS gene interaction also exhibited a gain-and-loss pattern during the legume evolution.To combat the constant challenges by pathogens, plants have evolved a sophisticated two-layered defense system, in which proteins encoded by disease RESISTANCE (R) genes serve to sense pathogen invasion signals and to elicit defense responses (Jones and Dangl, 2006; McDowell and Simon, 2006; Bent and Mackey, 2007). Over 140 R genes have been characterized from different flowering plants, which confer resistance to a large array of pathogens, including bacteria, fungi, oomycetes, viruses, and nematodes (Liu et al., 2007; Yang et al., 2013). Among these, about 80% belong to the NBS-LRR class, which encodes a central nucleotide-binding site (NBS) domain and a C-terminal leucine-rich repeat (LRR) domain. Based on whether their N termini are homologous to the Toll/Interleukin1 receptor (TIR), the angiosperm NBS-LRR genes are further divided into the TIR-NBS-LRR (TNL) subclass and the non-TIR-NBS-LRR (nTNL) subclass (Meyers et al., 1999; Bai et al., 2002; Cannon et al., 2002). The latter has been also called CC-NBS-LRR (CNL) subclass, since a coiled-coil (CC) structure is often detected at the N terminus (Meyers et al., 2003). Interestingly, a small group of nTNL genes have an N-terminal RPW8-like domain with a transmembrane region before the CC structure (Xiao et al., 2001). This group of RPW8-NBS-LRR (RNL) genes has been usually viewed as a specific lineage of CNLs (Bonardi et al., 2011; Collier et al., 2011); however, its real phylogenetic relationship with CNLs and TNLs requires further investigation.NBS-LRR genes have been surveyed in many sequenced genomes of flowering plants, including four monocots: rice (Oryza sativa), maize (Zea mays), sorghum (Sorghum bicolor), and Brachypodium distachyon; one basal eudicot: Nelumbo nucifera; two asterid species: potato (Solanum tuberosum) and tomato (Solanum lycopersicum); and 14 rosids: Vitis vinifera, Populus trichocarpa, Ricinus communis, Medicago truncatula, soybean (Glycine max), Lotus japonicus, Cucumis sativus, Cucumis melo, Citrullus lanatus, Gossypium raimondii, Carica papaya, Arabidopsis (Arabidopsis thaliana), Arabidopsis lyrata, and Brassica rapa (Bai et al., 2002; Meyers et al., 2003; Monosi et al., 2004; Zhou et al., 2004; Yang et al., 2006, 2008b; Ameline-Torregrosa et al., 2008; Mun et al., 2009; Porter et al., 2009; Chen et al., 2010; Li et al., 2010a, 2010b; Guo et al., 2011; Zhang et al., 2011; Jupe et al., 2012; Lozano et al., 2012; Luo et al., 2012; Tan and Wu, 2012; Andolfo et al., 2013; Jia et al., 2013; Lin et al., 2013; Wan et al., 2013; Wei et al., 2013; Wu et al., 2014). Variable numbers (from dozens to hundreds) of NBS-LRR genes were reported from these genomes, making one wonder: how did these genes evolve so variably during flowering plant speciation?Comparative genomic studies conducted in the available genome sequences of closely related species or subspecies revealed that a significant proportion of NBS-LRR genes are not shared. For example, 70 NBS-LRR genes between Arabidopsis and A. lyrata show the presence/absence of polymorphisms (Chen et al., 2010; Guo et al., 2011). Moreover, synteny analysis revealed that, among 363 NBS-LRR gene loci in indica (cv 93-11) and japonica (cv Nipponbare) rice, 124 loci exist in only one genome (Luo et al., 2012). Unequal crossover, homologous repair, and nonhomologous repair are the three ways that NBS-LRR gene deletions are caused in rice genomes (Luo et al., 2012).In many surveyed genomes, the majority of NBS-LRR genes are found in a clustered organization (physically close to each other), with the rest exhibited as singletons. Many clusters are homogenous, with only duplicated members occupying the same phylogenetic lineage, whereas heterogenous clusters comprise members from distantly related clades (Meyers et al., 2003). Leister (2004) defined three types of NBS gene duplications: local tandem, ectopic, and segmental duplications. Although a general agreement on the widespread occurrence of local tandem duplications can be reached by various genome survey studies, the relative importance of ectopic and segmental duplications has been seldom investigated since the earliest surveys of the Arabidopsis genome (Richly et al., 2002; Baumgarten et al., 2003; Meyers et al., 2003; McDowell and Simon, 2006).With more genomic data available in certain angiosperm families, NBS-LRR genes should be further investigated among phylogenetically distant species to fill the gaps in the understanding of their long-term evolutionary patterns. The legume family contains many economically important crop species, such as clover (Trifolium spp.), soybean, peanut (Arachis hypogaea), and common bean (Phaseolus vulgaris). Although these legumes are constantly threatened by various pathogens, only a few functional legume R genes have been characterized, and all of them belong to the NBS-LRR class (Ashfield et al., 2004; Hayes et al., 2004; Gao et al., 2005; Seo et al., 2006; Yang et al., 2008a; Meyer et al., 2009). Therefore, it would be interesting to investigate the NBS-LRR gene repertoire among different legume species. Here, we carried out a comprehensive analysis of NBS-LRR genes in four divergent legume genomes, M. truncatula, pigeon pea (Cajanus cajan), common bean, and soybean, which shared a common ancestor approximately 54 million years ago (MYA; Fig. 1; Lavin et al., 2005). Approximately 1,000 nTNL and 667 TNL subclass NBS-encoding genes were identified in our study. Their genomic distribution, organization modes, phylogenetic relationships, and syntenic patterns were examined to obtain insight into the long-term evolutionary patterns of NBS-LRR genes.Open in a separate windowFigure 1.The phylogenetic tree of four investigated legume species (M. truncatula, pigeon pea, common bean, and soybean). Two WGD events are indicated with triangles: one occurred approximately 59 MYA in the common ancestor of the four investigated legumes, and the other occurred approximately 13 MYA in the Glycine spp. lineage alone (Schmutz et al., 2010). The numbers at the branch nodes indicate divergence times (Lavin et al., 2005; Stefanovic et al., 2009). [See online article for color version of this figure.]     

The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-Related Protein Kinases Are Required for Elicitor-Induced Oxidative Burst and Immunity

Plant immunity is activated through complex and cross-talking transduction pathways that include a mitogen-activated protein kinase phosphorylation cascade. Here, we have investigated the role in immunity of the Arabidopsis (Arabidopsis thaliana) gene subfamily that encodes the mitogen-activated protein triple kinases indicated as ARABIDOPSIS NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-RELATED PROTEIN KINASE1 (ANP1), ANP2, and ANP3. For this study, we used representative danger signals (elicitors) belonging to the classes of the damage- and pathogen-associated molecular patterns, i.e. oligogalacturonides, linear fragments derived from the plant cell wall homogalacturonan, and the peptide elf18 derived from the bacterial elongation factor thermo-unstable. Analyses of single and double as well as conditional triple mutants show that ANPs are required for elicitor-triggered defense responses and protection against the necrotrophic fungus Botrytis cinerea. Notably, ANPs are also required for both the elicitor-induced oxidative burst and the transduction of the hydrogen peroxide signal but not for the inhibition of auxin-induced gene expression, indicating that this response can be uncoupled from the activation of defense responses. Our findings point to ANPs as key transduction elements that coordinate damage- and pathogen-associated molecular pattern-triggered immunity and orchestrate reactive oxygen species accumulation and signaling.Plants are continually exposed to microbial pathogens and, like animals, activate the innate immune system to respond properly and in a timely manner (Boller and He, 2009). Plants also rely on the structure of the cell wall that acts as a physical barrier against the microbial invasion (De Lorenzo et al., 2001). In their attempts to penetrate plant tissues, pathogens need to efficiently degrade the cell wall (Lionetti et al., 2010). Once the plant cell wall is breached, pathogens encounter the host plasma membrane, where pattern recognition receptors (PRRs) sense the presence of nonself molecules (pathogen-associated molecular patterns [PAMPs]) and activate the so-called PAMP-triggered immunity (PTI; Dodds and Rathjen, 2010). Endogenous molecules, released during infection or mechanical wounding and usually referred to as damage-associated molecular patterns (DAMPs), are also recognized by PRRs as danger signals and contribute to the activation of the plant immune response (Schwessinger and Ronald, 2012). Representative PAMPs are the peptides elf18 and flg22, derived from the bacterial elongation factor thermo-unstable (EF-Tu) and flagellin, respectively (Gómez-Gómez and Boller, 2000; Zipfel et al., 2006). Among the best characterized DAMPs are oligogalacturonides (OGs), linear molecules of 10 to about 16 α-1,4-d-galactopyranosyluronic acid residues released upon fragmentation of homogalacturonan, which is an important component of the plant cell wall (Ferrari et al., 2013). In Arabidopsis (Arabidopsis thaliana), elf18 and flg22 are recognized by the transmembrane leucine-rich repeat receptor kinases EF-TU RECEPTOR (EFR) and FLAGELLIN-SENSING2, respectively (Gómez-Gómez and Boller, 2000; Zipfel et al., 2006). OGs, instead, are recognized by the WALL-ASSOCIATED KINASE1 (WAK1), a receptor kinase containing epidermal growth factor-like repeats (Brutus et al., 2010).Activation of PRRs leads to a prompt induction of ion fluxes, an oxidative burst, and defense gene expression and to late responses such as callose deposition, seedling growth inhibition, and protection against further pathogen attack. An overlap but also some distinctive features exist between responses induced by PAMPs and DAMPs. For example, flg22 and OGs generate an extracellular oxidative burst mediated by RESPIRATORY BURST OXIDASE HOMOLOG D (RbohD) and induce protection against the necrotrophic fungus Botrytis cinerea independently of the ethylene, jasmonic acid, and salicylic acid pathways and of the RbohD-mediated production of reactive oxygen species (ROS; Zhang et al., 2007; Galletti et al., 2008). The inhibition of auxin responses is another feature shared by PAMPs and DAMPs (Savatin et al., 2011); in the case of OGs, the inhibition of auxin responses has been described as a true antagonism (Branca et al., 1988; Bellincampi et al., 1993; Savatin et al., 2011). On the other hand, microarray analyses indicate that late responses to the two classes of elicitors are considerably different (Denoux et al., 2008).In plants, as in animals, immunity is activated through complex and cross-talking transduction pathways that include a mitogen-activated protein (MAP) kinase (MAPK) phosphorylation cascade (Rodriguez et al., 2010). A MAPK cascade consists of a core module of three kinases that perform sequential phosphorylation reactions: a MAP kinase kinase kinase (MAP3K) activates, by phosphorylation, a MAP kinase kinase (MAP2K), which activates a MAPK. Sixty MAP3Ks, 10 MAP2Ks, and 20 MAPKs are encoded by the Arabidopsis genome (Ichimura et al., 2002), leading to a complexity that hampers the characterization of this transduction system. Three immune-related MAPK modules have been identified. A module comprising the MAP3K MEKK1, the MAP2Ks MKK1 and MKK2, and the MAPK MPK4 (MEKK1/MKK1-MKK2/MPK4) negatively controls defense responses (Kong et al., 2012; Rasmussen et al., 2012) by negatively regulating the expression of the MAP3K MEKK2, which triggers a salicylate (SA)-dependent autoimmunity response when the cascade is compromised (Berriri et al., 2012; Su et al., 2013). Two other modules, MEKK1-MAPKKKα/MKK4-MKK5/MPK3-MPK6 (Ren et al., 2008) and MKK9/MPK3-MPK6 (Xu et al., 2008), mediate the activation of defense responses, including the synthesis of ethylene and camalexin, i.e. a phytoalexin with antimicrobial activity. The only MAPK elements shown so far to participate in the response to DAMPs are the Arabidopsis MPK3 and MPK6. Both are phosphorylated within minutes upon elicitation with OGs and flg22, but only MPK6 is required for full elicitor-induced up-regulation of defense genes and protection against B. cinerea (Galletti et al., 2011).The subfamily of MAP3Ks indicated as ARABIDOPSIS NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1 (NPK1)-RELATED PROTEIN KINASEs (ANPs) includes three members, ANP1, ANP2, and ANP3, that were initially identified for their homology with the tobacco (Nicotiana tabacum) NPK1 (Jouannic et al., 1999; Sasabe and Machida, 2012). NPK1 regulates cytokinesis (Nakashima et al., 1998) as well as the effector-triggered immunity and development in Nicotiana benthamiana (Jin et al., 2002). ANPs have also been reported to be involved in the inhibition of auxin-induced gene expression. Constitutively active forms of ANPs, obtained by deletion of the C-terminal regulatory domain (ΔANPs) and expressed in protoplasts, negatively regulate the activity of the auxin-inducible soybean (Glycine max) GRETCHEN HAGEN3 promoter and activate the hydrogen peroxide signaling pathway (Kovtun et al., 2000). Therefore, ANPs have been proposed as a molecular link between oxidative stress and auxin signal transduction. However, we have previously shown that double knockout (KO) anp mutants exhibit a normal auxin/OG antagonism (Savatin et al., 2011), thus providing no support to this conclusion. Our work left unanswered whether this was due to the presence of a functional third ANP member or to a lack of involvement of ANPs in elicitor/auxin antagonism and, in general, in the response to OGs, because a main role of ANP1 in response to flg22 had been previously ruled out (Asai et al., 2002).The anp2 anp3 double mutant displays developmental defects related to cytokinesis as well as up-regulation of stress-related genes, while an anp triple KO mutant was never obtained, probably because ANPs are essential for plant development (Krysan et al., 2002). The phenotype of the anp2 anp3 mutant is similar to that of mpk4 mutant, suggesting that ANPs and MPK4 may be part of the same transduction pathway and act as negative regulators of defense responses (Beck et al., 2011).We show here that ANP genes are not involved in elicitor-auxin antagonism but are required for DAMP and PAMP signal transduction. Single and double mutants as well as conditional triple mutants, which were generated in this work, are defective in defense responses to both OGs and elf18. Notably, ANPs are required for both elicitor-induced generation of ROS and response to ROS. Our study points to ANPs as key regulators of plant immunity.     

Two Mitogen-Activated Protein Kinases,MPK3 and MPK6, Are Required for Funicular Guidance of Pollen Tubes in Arabidopsis

Double fertilization in flowering plants requires the delivery of two immotile sperm cells to the female gametes by a pollen tube, which perceives guidance cues, modifies its tip growth direction, and eventually enters the micropyle of the ovule. In spite of the recent progress, so far, little is known about the signaling events in pollen tubes in response to the guidance cues. Here, we show that MPK3 and MPK6, two Arabidopsis (Arabidopsis thaliana) mitogen-activated protein kinases, mediate the guidance response in pollen tubes. Genetic analysis revealed that mpk3 mpk6 double mutant pollen has reduced transmission. However, direct observation of mpk3 mpk6 mutant pollen phenotype was hampered by the embryo lethality of double homozygous mpk3^–/– mpk6^–/– plants. Utilizing a fluorescent reporter-tagged complementation method, we showed that the mpk3 mpk6 mutant pollen had normal pollen tube growth but impaired pollen tube guidance. In vivo pollination assays revealed that the mpk3 mpk6 mutant pollen tubes were defective in the funicular guidance phase. By contrast, semi-in vitro guidance assay showed that the micropylar guidance of the double mutant pollen tube was normal. Our results provide direct evidence to support that the funicular guidance phase of the pollen tube requires an in vivo signaling mechanism distinct from the micropyle guidance. Moreover, our finding opened up the possibility that the MPK3/MPK6 signaling pathway may link common signaling networks in plant stress response and pollen-pistil interaction.In flowering plants, successful fertilization is dependent on extensive cell-cell communication between male and female gametophytes. After landing on a compatible stigma surface, a mature pollen grain germinates to form a pollen tube, which penetrates the stigma, perceives guidance cues along the growth path, and modifies its tip growth direction toward the ovule (Hülskamp et al., 1995). In Arabidopsis (Arabidopsis thaliana), the pollen tube guidance can be divided into two phases: funicular guidance, in which the pollen tube emerges from the septum and proceeds to a funiculus, and micropylar guidance, in which the pollen tube grows toward and enters the micropyle of an ovule (Hülskamp et al., 1995).In pollen tube, it is believed that receptors on the tube tip perceive various guidance cues and regulate downstream signaling pathways to modify tip reorientation toward the ovule (Higashiyama, 2010; Takeuchi and Higashiyama, 2011). Two receptor-like kinase genes, Lost In Pollen tube guidance1 (LIP1) and LIP2, are involved in guidance control of pollen tubes. LIP1 and LIP2 were anchored to the membrane in the pollen tube tip region via palmitoylation, which was essential for their guidance control (Liu et al., 2013). Therefore, LIP1 and LIP2 are the essential components of the receptor complex in micropylar guidance. The Glu receptor-like channels facilitate Ca²⁺ influx across the plasma membrane and regulate pollen tube growth and morphogenesis (Michard et al., 2011). This interesting work revealed that there is a signaling mechanism between the male gametophyte and pistil tissue that is similar to the amino acid-mediated communication in animal nervous systems (Michard et al., 2011). Recent findings also highlight the importance of the endoplasmic reticulum (ER), ion homeostasis, and protein processing in pollen tube guidance (Li et al., 2011; Lu et al., 2011; Li and Yang, 2012). Two pollen-expressed cation proton exchangers (CHXs), CHX21 and CHX23, were reported to mediate K⁺ transport in ER and are essential for the pollen tube to respond to directional signals from the ovule in Arabidopsis (Lu et al., 2011). POLLEN DEFECTIVE IN GUIDANCE1 plays an important role in micropylar guidance in pollen tube (Li et al., 2011). It is an ER luminal protein involved in ER protein retention and interacts with a luminal chaperone involved in Ca²⁺ homeostasis and ER quality control (Li et al., 2011). Therefore, the ER quality control is likely an important mechanism in surveillance of signaling factors in pollen tube guidance (Li and Yang, 2012).In spite of the recent progresses, so far, little is known about the cytoplasmic signaling events in pollen tubes in response to the guidance cues. Mitogen-activated protein kinase (MAPK, or MPK) cascades are conserved signaling pathways that respond to extracellular stimuli and regulate various cellular activities. In Arabidopsis, MPK3 and MPK6 are induced by various biotic and abiotic stresses and collaboratively play important roles in defense response and plant development (Zhang, 2008). Here, we show that MPK3 and MPK6 are also critical to pollen tube guidance. Utilizing a fluorescent reporter-tagged complementation method, we demonstrated that mpk3 mpk6 pollen was defective in pollen tube guidance at the funicular guidance phase. Intriguingly, the micropylar guidance of mpk3 mpk6 pollen tube is not affected.     

Fungal Endopolygalacturonases Are Recognized as Microbe-Associated Molecular Patterns by the Arabidopsis Receptor-Like Protein RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1

Plants perceive microbial invaders using pattern recognition receptors that recognize microbe-associated molecular patterns. In this study, we identified RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1 (RBPG1), an Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like protein, AtRLP42, that recognizes fungal endopolygalacturonases (PGs) and acts as a novel microbe-associated molecular pattern receptor. RBPG1 recognizes several PGs from the plant pathogen Botrytis cinerea as well as one from the saprotroph Aspergillus niger. Infiltration of B. cinerea PGs into Arabidopsis accession Columbia induced a necrotic response, whereas accession Brno (Br-0) showed no symptoms. A map-based cloning strategy, combined with comparative and functional genomics, led to the identification of the Columbia RBPG1 gene and showed that this gene is essential for the responsiveness of Arabidopsis to the PGs. Transformation of RBPG1 into accession Br-0 resulted in a gain of PG responsiveness. Transgenic Br-0 plants expressing RBPG1 were equally susceptible as the recipient Br-0 to the necrotroph B. cinerea and to the biotroph Hyaloperonospora arabidopsidis. Pretreating leaves of the transgenic plants with a PG resulted in increased resistance to H. arabidopsidis. Coimmunoprecipitation experiments demonstrated that RBPG1 and PG form a complex in Nicotiana benthamiana, which also involves the Arabidopsis leucine-rich repeat receptor-like protein SOBIR1 (for SUPPRESSOR OF BIR1). sobir1 mutant plants did not induce necrosis in response to PGs and were compromised in PG-induced resistance to H. arabidopsidis.Microbe-associated molecular patterns (MAMPs) are molecular signatures of entire groups of microbes and have key roles in activation of the defense response in plants (Jones and Dangl, 2006; Boller and Felix, 2009). Well-characterized proteinaceous MAMPs are bacterial flagellin, Elongation Factor Tu (EF-Tu), and Ax21, fungal xylanase, and oomycete pep13, an epitope of a secreted transglutaminase (Boller and Felix, 2009; Monaghan and Zipfel, 2012). Plants recognize MAMPs by means of pattern recognition receptors (PRRs), comprising a group of leucine-rich repeat (LRR) receptor-like kinases (RLKs) or LRR receptor-like proteins (RLPs) located in the plasma membrane (Greeff et al., 2012; Monaghan and Zipfel, 2012). The LRR-RLKs FLS2 and EFR recognize flg22 (the 22-amino acid eliciting epitope from the conserved flagellin domain) and elf18/elf26 (peptides derived from the N terminus of translation elongation factor EF-Tu), respectively (Gómez-Gómez and Boller, 2000; Kunze et al., 2004; Chinchilla et al., 2006; Zipfel et al., 2006). The fungal protein ethylene-inducing xylanase (EIX) is recognized by the tomato (Solanum lycopersicum) LRR-RLPs LeEIX1 and LeEIX2, of which only the latter mediates a necrotic response (Ron and Avni, 2004).BRI1-ASSOCIATED KINASE1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE3 (BAK1/SERK3) is an LRR-RLK acting as a common component in many RLK signaling complexes (Monaghan and Zipfel, 2012). Although it was originally identified as a protein that interacts with the brassinosteroid receptor BRI1 (Li et al., 2002; Nam and Li, 2002), BAK1 also forms ligand-induced complexes with FLS2 and EFR and contributes to disease resistance against the pathogens Pseudomonas syringae, Hyaloperonospora arabidopsidis (Hpa), and Phytophthora infestans (Chinchilla et al., 2007; Heese et al., 2007; Chaparro-Garcia et al., 2011; Roux et al., 2011). Tomato BAK1 interacts in a ligand-independent manner with LeEIX1 but not with LeEIX2, and the BAK1-LeEIX1 interaction is required for the ability of LeEIX1 to attenuate the signaling of LeEIX2 (Bar et al., 2010). BAK1 has also been shown to interact with another LRR-RLK, BAK1-INTERACTING RECEPTOR-LIKE KINASE1 (BIR1). A bir1 mutant showed extensive cell death, activation of constitutive defense responses, and impairment in the activation of the mitogen-activated protein kinase MPK4 (Gao et al., 2009). sobir1 (for suppressor of BIR1) mutants suppress BIR1 phenotypes, and overexpression of SOBIR1 triggers cell death and defense responses (Gao et al., 2009). SOBIR1 does not physically interact with BIR1, suggesting that SOBIR1 mediates an alternative signal transduction route. A recent study showed that the tomato SOBIR1 interacts with RLPs and is required for RLP-mediated disease resistance (Liebrand et al., 2013).Endopolygalacturonases (PGs) are a class of pectinases that hydrolyze the homogalacturonan domain of pectic polysaccharides (van den Brink and de Vries, 2011). Secreted PGs are able to cause cell wall decomposition and tissue maceration and thereby act as virulence factors in several fungal pathogens, such as Aspergillus flavus, Claviceps purpurea, and Alternaria citri (Shieh et al., 1997; Isshiki et al., 2001; Oeser et al., 2002). The most extensively studied PGs from fungal plant pathogens are those of Botrytis cinerea (for review, see Zhang and van Kan, 2013b), a necrotrophic broad-host-range pathogen that contains six PG genes (designated Bcpg1–Bcpg6) in its genome (Wubben et al., 1999). Deletion of either Bcpg1 or Bcpg2 resulted in a strong reduction in virulence on tomato and broad bean (Vicia faba) leaves (ten Have et al., 1998; Kars et al., 2005), presumably because the enzymes have a detrimental effect on the integrity of host cell walls and tissues. Indeed, infiltrating BcPG2 into broad bean leaves or transient expression of BcPG2 in Nicotiana benthamiana led to tissue collapse and necrosis, and the necrotic response was abolished when the catalytic domain of the PG was mutated (Kars et al., 2005; Joubert et al., 2007). By contrast, Poinssot et al. (2003) reported that BcPG1 can activate plant defense responses in grapevine (Vitis vinifera) cell suspensions independently of its enzymatic activity, suggesting that the protein itself might be recognized by plant cells as an elicitor. These studies with seemingly opposing conclusions were conducted with different isozymes on distinct cell types of different plant species. Thus, it remained inconclusive whether plant responses observed after exposure to PGs are due to the structural damage resulting from pectin hydrolysis or to recognition of the protein as a MAMP (followed by downstream signaling responses).The degradation of pectin by PGs leads to the release of oligogalacturonides (OGAs), which may activate a variety of defense responses (Prade et al., 1999; D’Ovidio et al., 2004). OGAs act as damage-associated molecular patterns (DAMPs) via their perception by the cell wall-associated receptor Wall-Associated Kinase1 (WAK1; Brutus et al., 2010). Overexpression of WAK1 in Arabidopsis (Arabidopsis thaliana) enhances resistance to B. cinerea (Brutus et al., 2010).Here, we describe the occurrence of natural variation among Arabidopsis accessions in responsiveness to fungal PGs. Two accessions that strongly differed in their response to PGs were selected for further analysis. Cloning and functional characterization demonstrated that the gene RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1 (RBPG1) encodes an LRR-RLP. Finally, we demonstrate that the LRR-RLK SUPPRESSOR OF BIR1 (SOBIR1) interacts with RBPG1 and is essential for responsiveness to fungal PGs.     

Temperature Responses of C4 Photosynthesis: Biochemical Analysis of Rubisco,Phosphoenolpyruvate Carboxylase,and Carbonic Anhydrase in Setaria viridis

The photosynthetic assimilation of CO₂ in C₄ plants is potentially limited by the enzymatic rates of Rubisco, phosphoenolpyruvate carboxylase (PEPc), and carbonic anhydrase (CA). Therefore, the activity and kinetic properties of these enzymes are needed to accurately parameterize C₄ biochemical models of leaf CO₂ exchange in response to changes in CO₂ availability and temperature. There are currently no published temperature responses of both Rubisco carboxylation and oxygenation kinetics from a C₄ plant, nor are there known measurements of the temperature dependency of the PEPc Michaelis-Menten constant for its substrate HCO₃⁻, and there is little information on the temperature response of plant CA activity. Here, we used membrane inlet mass spectrometry to measure the temperature responses of Rubisco carboxylation and oxygenation kinetics, PEPc carboxylation kinetics, and the activity and first-order rate constant for the CA hydration reaction from 10°C to 40°C using crude leaf extracts from the C₄ plant Setaria viridis. The temperature dependencies of Rubisco, PEPc, and CA kinetic parameters are provided. These findings describe a new method for the investigation of PEPc kinetics, suggest an HCO₃⁻ limitation imposed by CA, and show similarities between the Rubisco temperature responses of previously measured C₃ species and the C₄ plant S. viridis.Biochemical models of photosynthesis are often used to predict the effect of environmental conditions on net rates of leaf CO₂ assimilation (Farquhar et al., 1980; von Caemmerer, 2000, 2013; Walker et al., 2013). With climate change, there is increased interest in modeling and understanding the effects of changes in temperature and CO₂ concentration on photosynthesis. The biochemical models of photosynthesis are primarily driven by the kinetic properties of the enzyme Rubisco, the primary carboxylating enzyme of the C₃ photosynthetic pathway, catalyzing the reaction of ribulose-1,5-bisphosphate (RuBP) with either CO₂ or oxygen. However, the CO₂-concentrating mechanism in C₄ photosynthesis utilizes carbonic anhydrase (CA) to help maintain the chemical equilibrium of CO₂ with HCO₃⁻ and phosphoenolpyruvate carboxylase (PEPc) to catalyze the carboxylation of phosphoenolpyruvate (PEP) with HCO₃⁻. These reactions ultimately provide the elevated levels of CO₂ to the compartmentalized Rubisco (Edwards and Walker, 1983). In C₄ plants, it has been demonstrated that PEPc, Rubisco, and CA can limit rates of CO₂ assimilation and influence the efficiency of the CO₂-concentrating mechanism (von Caemmerer, 2000; von Caemmerer et al., 2004; Studer et al., 2014). Therefore, accurate modeling of leaf photosynthesis in C₄ plants in response to future climatic conditions will require temperature parameterizations of Rubisco, PEPc, and CA kinetics from C₄ species.Modeling C₄ photosynthesis relies on the parameterization of both PEPc and Rubisco kinetics, making it more complex than for C₃ photosynthesis (Berry and Farquhar, 1978; von Caemmerer, 2000). However, the activity of CA is not included in these models, as it is assumed to be nonlimiting under most conditions (Berry and Farquhar, 1978; von Caemmerer, 2000). This assumption is implemented by modeling PEPc kinetics as a function of CO₂ partial pressure (pCO₂) and not HCO₃⁻ concentration, assuming CO₂ and HCO₃⁻ are in chemical equilibrium. However, there are questions regarding the amount of CA activity needed to sustain rates of C₄ photosynthesis and if CO₂ and HCO₃⁻ are in equilibrium (von Caemmerer et al., 2004; Studer et al., 2014).The most common steady-state biochemical models of photosynthesis are derived from the Michaelis-Menten models of enzyme activity (von Caemmerer, 2000), which are driven by the V_max and the K_m. Both of these parameters need to be further described by their temperature responses to be used to model photosynthesis in response to temperature. However, the temperature response of plant CA activity has not been completed above 17°C, and there is no known measured temperature response of K_m HCO₃⁻ for PEPc (K_P). Alternatively, Rubisco has been well studied, and there are consistent differences in kinetic values between C₃ and C₄ species at 25°C (von Caemmerer and Quick, 2000; Kubien et al., 2008), but the temperature responses, including both carboxylation and oxygenation reactions, have only been performed in C₃ species (Badger and Collatz, 1977; Jordan and Ogren, 1984; Bernacchi et al., 2001, 2002; Walker et al., 2013).Here, we present the temperature dependency of Rubisco carboxylation and oxygenation reactions, PEPc kinetics for HCO₃⁻, and CA hydration from 10°C to 40°C from the C₄ species Setaria viridis (succession no., A-010) measured using membrane inlet mass spectrometry. Generally, the 25°C values of the Rubisco parameters were similar to previous measurements of C₄ species. The temperature response of the maximum rate of Rubisco carboxylation (V_cmax) was high compared with most previous measurements from both C₃ and C₄ species, and the temperature response of the K_m for oxygenation (K_O) was low compared with most previously measured species. Taken together, the modeled temperature responses of Rubisco activity in S. viridis were similar to the previously reported temperature responses of some C₃ species. Additionally, the temperature response of the maximum rate of PEPc carboxylation (V_pmax) was similar to previous measurements. However, the temperature response of K_P was lower than what has been predicted (Chen et al., 1994). For CA, deactivation of the hydration activity was observed above 25°C. Additionally, models of CA and PEPc show that CA activity limits HCO₃⁻ availability to PEPc above 15°C, suggesting that CA limits PEP carboxylation rates in S. viridis when compared with the assumption that CO₂ and HCO₃⁻ are in full chemical equilibrium.     

Engineering Monolignol p-Coumarate Conjugates into Poplar and Arabidopsis Lignins

Lignin acylation, the decoration of hydroxyls on lignin structural units with acyl groups, is common in many plant species. Monocot lignins are decorated with p-coumarates by the polymerization of monolignol p-coumarate conjugates. The acyltransferase involved in the formation of these conjugates has been identified in a number of model monocot species, but the effect of monolignol p-coumarate conjugates on lignification and plant growth and development has not yet been examined in plants that do not inherently possess p-coumarates on their lignins. The rice (Oryza sativa) p-COUMAROYL-Coenzyme A MONOLIGNOL TRANSFERASE gene was introduced into two eudicots, Arabidopsis (Arabidopsis thaliana) and poplar (Populus alba × grandidentata), and a series of analytical methods was used to show the incorporation of the ensuing monolignol p-coumarate conjugates into the lignin of these plants. In poplar, specifically, the addition of these conjugates did not occur at the expense of the naturally incorporated monolignol p-hydroxybenzoates. Plants expressing the p-COUMAROYL-Coenzyme A MONOLIGNOL TRANSFERASE transgene can therefore produce monolignol p-coumarate conjugates essentially without competing with the formation of other acylated monolignols and without drastically impacting normal monolignol production.Lignification of plant cell walls prototypically involves the polymerization of the monolignols (MLs), p-coumaryl alcohol, coniferyl alcohol (CA), and sinapyl alcohol (SA), predominantly by stepwise radical coupling of each monomer to the phenolic end of the growing polymer (Sarkanen and Ludwig, 1971; Boerjan et al., 2003; Ralph et al., 2004). The contribution of various MLs to the lignins depends on plant species, cell type, plant tissue, and tissue age. Although the majority of the lignin polymer is derived from these three MLs, the lignification process has a high degree of metabolic plasticity (Boerjan et al., 2003; Ralph et al., 2004; Ralph, 2007; Vanholme et al., 2012). Of particular interest are ML conjugates in which the ester group can be acetate (Ac; Sarkanen et al., 1967; Ralph, 1996; Ralph and Lu, 1998; Del Río et al., 2007; del Río et al., 2008; Martínez et al., 2008), p-hydroxybenzoate (pBz; Venverloo, 1971; Monties and Lapierre, 1981; Landucci et al., 1992; Tomimura, 1992a, 1992b; Hibino et al., 1994; Sun et al., 1999; Kuroda et al., 2001; Lu et al., 2004, 2015; Morreel et al., 2004; Rencoret et al., 2013), p-coumarate (pCA; Monties and Lapierre, 1981; Ralph et al., 1994; Crestini and Argyropoulos, 1997; del Río et al., 2008, 2012a, 2012b; Withers et al., 2012; Rencoret et al., 2013; Petrik et al., 2014), or ferulate (FA; Grabber et al., 2008; Ralph, 2010; Wilkerson et al., 2014). In all cases, the MLs are acylated before polymerization as proven by the presence in the lignins of unique β-β coupling products that only arise when one or both of the MLs are acylated, preventing the formation of the typical resinols from internal trapping of the quinone methide intermediates by the γ-OH (Lu and Ralph, 2002, 2008; Del Río et al., 2007; Lu et al., 2015).The BAHD acyltransferase, FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT), was recently identified in Angelica sinensis and transformed into poplar (Populus alba × grandidentata), which naturally incorporates other acylated MLs, namely ML-pBz conjugates, into its lignin (Wilkerson et al., 2014). Plants that incorporate ML-FAs into their lignins have the potential to be particularly important economically, because their lignin backbones are permeated with readily cleavable ester bonds, facilitating lignin breakdown and removal under alkaline pretreatment conditions. Determining the extent to which ML-FAs are incorporated into the lignin polymer is, however, extremely difficult because of the diversity of products generated during the polymerization events, which is described in the supplemental information in Wilkerson et al., 2014.There is currently only one technique, derivatization followed by reductive cleavage (DFRC), that can release diagnostic chemical marker compounds from lignins containing ML-FAs (Lu and Ralph, 2014; Wilkerson et al., 2014). The DFRC method selectively cleaves β-ethers while leaving ester linkages intact. This technique was recently used to show that ML-FA conjugates are fully incorporated into the lignin of the FMT poplar (Wilkerson et al., 2014), but the extent of incorporation, the spatial distribution, the exact mechanism of delivery to the developing cell wall, and the efficiency of incorporation remain largely unknown.The biological role of pCA in lignin has been highly speculative. It is hypothesized that the pCA moieties may function as a radical sensitizer (Takahama and Oniki, 1996, 1997; Takahama et al., 1996; Ralph et al., 2004; Hatfield et al., 2008; Ralph, 2010). Peroxidases and/or laccases readily oxidize pCA to a radical but are poor oxidizers for SA. Free radicals of pCA readily undergo radical transfer to SA, which in turn, forms a homodimer or couples to the end of a growing polymer chain. Conjugating pCA to an ML, like SA, to form SA-pCA, the most prevalent ML-pCA conjugate in grasses, creates a compound with a built-in radical sensitizer that can participate in the polymerization event. The prevalence of these conjugates in potential biofuel crops and the impact that these ester-linked conjugates have on the lignin polymer during pretreatment and downstream fermentation processes have driven the search to find the genes and their enzymes responsible for acylating MLs in monocots (Withers et al., 2012; Marita et al., 2014; Petrik et al., 2014; Wilkerson et al., 2014).In rice (Oryza sativa), enzymes have been characterized that function specifically in the addition of pCA onto hemicelluloses (Bartley et al., 2013) or lignin (Withers et al., 2012; Petrik et al., 2014). The p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (PMT) was identified as one of many grass-specific BAHD acyltransferases produced by rice and found to coexpress with many ML biosynthetic enzymes (Withers et al., 2012). The enzyme preferentially forms a γ-ester through its specificity toward p-coumaroyl-CoA and an ML, and has kinetic efficiency with p-coumaryl alcohol > SA > CA. In most grasses, the PMT enzyme predominantly produces SA-pCA conjugates that are then incorporated into the lignin polymer (Petrik et al., 2014).To test the role of PMT during cell wall lignification, genetic manipulation of PMT genes has been performed in Brachypodium distachyon and maize (Zea mays), two model monocots. The suppression and overexpression of a BdPMT revealed the PMT to be involved only in the acylation of MLs before polymerization and not in the acylation of hemicelluloses (Petrik et al., 2014). RNA interference-mediated suppression of BdPMT resulted in decreased incorporation of ML-pCA conjugates into the cell wall without adversely affecting growth, height, or digestibility of the mature plants. Even deleterious mutations in the BdPMT gene, which resulted in a complete absence of pCA-acylating B. distachyon lignins, did not affect plant growth or development (Petrik et al., 2014). The arabinose-bound FA and pCA levels remained virtually unchanged in the PMT-misregulated plants, illustrating the specificity of the PMT enzyme for the p-coumaroyl-CoA substrate and its ML acylation. The PMT enzyme identified in maize (pCAT = ZmPMT) also displayed the highest catalytic efficiency with p-coumaroyl-CoA and SA as substrates (Marita et al., 2014). RNAi-mediated suppression of ZmPMT also resulted in decreased production of the ML conjugates. The effect on the lignin polymer when introducing PMT into plants that do not normally express a homologous enzyme is, however, unknown.pCAs, because they favor radical transfer over radical coupling, are overwhelmingly seen as free-phenolic pendant entities on the lignin polymer (Ralph et al., 1994; Ralph, 2010). As a result, the pCA itself can be completely quantified by simple saponification. The units to which the pCA is attached are, like their normal ML-derived counterparts, not fully releasable from lignin as identifiable monomers (during degradative reactions), but the pCA’s terminal location makes p-coumaroylated units more readily releasable and detectable than if they participated in lignification (as FAs do). Examining the effect of PMT and its resulting conjugates on lignification in plants that do not naturally produce such conjugates will contribute to our understanding of the role of PMT in lignification in general.In this study, we aimed to assess the ability of the model eudicot plants Arabidopsis (Arabidopsis thaliana) and poplar, neither of which naturally produces ML-pCA conjugates, to express a PMT gene and incorporate these novel conjugates into their cell wall lignins. We also investigated the effect that the introduction of PMT has on the native levels of ML-pBz conjugates in poplar lignin. Various analytical techniques were optimized and used to examine the cell walls of the transgenic plants for pCA conjugates and determine whether they were specifically incorporated into the lignin polymer in the cell wall.     

The Leucine-Rich Repeat Receptor-Like Kinase BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 and the Cytochrome P450 PHYTOALEXIN DEFICIENT3 Contribute to Innate Immunity to Aphids in Arabidopsis

The importance of pathogen-associated molecular pattern-triggered immunity (PTI) against microbial pathogens has been recently demonstrated. However, it is currently unclear if this layer of immunity mediated by surface-localized pattern recognition receptors (PRRs) also plays a role in basal resistance to insects, such as aphids. Here, we show that PTI is an important component of plant innate immunity to insects. Extract of the green peach aphid (GPA; Myzus persicae) triggers responses characteristic of PTI in Arabidopsis (Arabidopsis thaliana). Two separate eliciting GPA-derived fractions trigger induced resistance to GPA that is dependent on the leucine-rich repeat receptor-like kinase BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1)/SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE3, which is a key regulator of several leucine-rich repeat-containing PRRs. BAK1 is required for GPA elicitor-mediated induction of reactive oxygen species and callose deposition. Arabidopsis bak1 mutant plants are also compromised in immunity to the pea aphid (Acyrthosiphon pisum), for which Arabidopsis is normally a nonhost. Aphid-derived elicitors induce expression of PHYTOALEXIN DEFICIENT3 (PAD3), a key cytochrome P450 involved in the biosynthesis of camalexin, which is a major Arabidopsis phytoalexin that is toxic to GPA. PAD3 is also required for induced resistance to GPA, independently of BAK1 and reactive oxygen species production. Our results reveal that plant innate immunity to insects may involve early perception of elicitors by cell surface-localized PRRs, leading to subsequent downstream immune signaling.Close to a million insect species have so far been described, and nearly one-half of them feed on plants (Wu and Baldwin, 2010). Within these plant-feeding insects, most feed on a few related plant species, with only 10% feeding upon multiple plant families (Schoonhoven et al., 2005). Plant defense to insects include several layers (Bos and Hogenhout, 2011; Hogenhout and Bos, 2011). Physical barriers, volatile cues, and composition of secondary metabolites of plants are important components that determine insect host choice (Howe and Jander, 2008; Bruce and Pickett, 2011). In addition, plants induce a variety of plant defense responses upon perception of herbivore oral secretions (OS), saliva, and eggs (De Vos and Jander, 2009; Bruessow et al., 2010; Ma et al., 2010; Wu and Baldwin, 2010). These responses may provide full protection against the majority of insect herbivores, and insects that are able to colonize specific plant species likely produce effectors in their saliva or during egg laying that suppress these induced defense responses (Bos and Hogenhout, 2011; Hogenhout and Bos, 2011; Pitino and Hogenhout, 2013).Aphids are sap-feeding insects of the order Hemiptera and are among the most destructive pests in agriculture, particularly in temperate regions (Blackman and Eastop, 2000). More than 4,000 aphid species in 10 families are known (Dixon, 1998). Most aphid species are specialists and use one or a few closely related plant species within one family as host for feeding and reproduction. Examples are pea aphid (Acyrthosiphon pisum), cabbage aphid (Brevicoryne brassicae), and English grain aphid (Sitobion avenae) that colonize plant species within the legumes (family Fabaceae), brassicas (Brassicaceae), and grasses (Gramineae), respectively. The green peach aphid (GPA; Myzus persicae) is one of few aphid species with a broad host range and can colonize hundreds of plants species in over 40 plant families, including brassicas (Blackman and Eastop, 2000). Aphids possess mouthparts composed of stylets that navigate to the plant vascular system, predominantly the phloem, for long-term feeding. However, before establishing a long-term feeding site, these insects display a host selection behavior by probing the upper leaf cell layers with their stylets, a behavior seen on host and nonhost plants of the aphid (Nam and Hardie, 2012). When the plant is judged unsuitable, the aphid takes off to find an alternative plant host. It is not yet clear what happens in the initial stages of insect interactions with plants.Plants sense microbial organisms (including bacteria, fungi, and oomycetes) through perception of conserved molecules, named microbe-associated molecular patterns and pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) to induce the first stage of plant immunity, termed PAMP-triggered immunity (PTI). PTI is effective against the majority of plant pathogens. Bacterial and fungal PAMPs characterized so far include bacterial flagellin (or its derived peptide flg22), bacterial elongation factor (EF)-Tu (or its derived peptide elf18), bacterial lipopolysaccharides and bacterial cold shock protein, chitin oligosaccharides, and the oomycete elicitin INF1 (Boller and Felix, 2009)Plant PRRs are either receptor-like kinases (RLKs) or receptor-like proteins. Most leucine-rich repeat (LRR)-type PRRs associate with and rely for their function on the small regulatory LRR-RLK BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1)/SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE3 (SERK3; Monaghan and Zipfel, 2012). For example, in Arabidopsis (Arabidopsis thaliana), flg22 and elf18 bind to the LRR-RLKs FLAGELLIN SENSITIVE2 (FLS2) and EF-TU RECEPTOR (EFR), respectively, leading to a quasi-instant association with BAK1 (Gómez-Gómez and Boller, 2000; Zipfel et al., 2006; Chinchilla et al., 2007; Heese et al., 2007; Schulze et al., 2010; Roux et al., 2011; Sun et al., 2013). BAK1 is required for optimal downstream immune signaling events, such as mitogen-activated protein kinase (MAPK) activation, reactive oxygen species (ROS) bursts, callose depositions, induction of immune genes, and induced resistance. Similarly, BAK1 is a positive regulator of innate immune responses triggered by the Arabidopsis LRR-RLKs PLANT ELICITOR PEPTIDE1 RECEPTOR1 (PEPR1) and PEPR2 that bind the Arabidopsis-derived damage-associated molecular pattern A. thaliana Peptide1 (AtPep1; Krol et al., 2010; Postel et al., 2010; Roux et al., 2011) and by the tomato (Solanum lycopersicum) LRR receptor-like protein Ve1 that recognizes Ave1 derived from Verticillium spp. (Fradin et al., 2009; de Jonge et al., 2012). Consistent with the role of BAK1 downstream of numerous PRRs, BAK1 is required for full immunity to a number of bacterial, fungal, oomycete, and viral pathogens (Heese et al., 2007; Kemmerling et al., 2007; Fradin et al., 2009; Chaparro-Garcia et al., 2011; Roux et al., 2011; Kørner et al., 2013).Notably, it has been recently shown that the ortholog of BAK1 in Nicotiana attenuata regulates the induction of jasmonic acid (JA) accumulation upon herbivory (Yang et al., 2011a). However, immunity to insects was not affected when BAK1 was silenced, and the observed effect on JA accumulation may be due to an indirect effect on brassinosteroid (BR) responses, for which BAK1 is also an important positive regulator (Li et al., 2002; Nam and Li, 2002). Therefore, it is currently unclear if BAK1 is involved in the early recognition of insect-derived elicitors leading to immunity.We discovered that the key regulatory LRR-RLK BAK1 participates in plant defense to an insect herbivore. We found that extracts of GPA trigger plant defense responses in Arabidopsis that are characteristic of PTI. Arabidopsis bak1 mutant plants are compromised in defense to GPA, which colonizes Arabidopsis, and to pea aphid, for which Arabidopsis is a nonhost. BAK1 is required for ROS bursts, callose deposition, and induced resistance in Arabidopsis upon perception of aphid-derived elicitors. One of the defense genes induced by GPA-derived extracts encodes PHYTOALEXIN DEFICIENT3 (PAD3), a cytochrome P450 that catalyzes the conversion of dihydrocamalexic acid to camalexin, which is a major Arabidopsis phytoalexin that is toxic to GPA (Kettles et al., 2013). PAD3 expression is required for Arabidopsis-induced resistance to GPA, independently of BAK1 and ROS. Our results provide evidence that innate immunity to insect herbivores may rely on the early perception of elicitors by cell surface-localized PRR.     

Cytochrome P450 CYP89A9 Is Involved in the Formation of Major Chlorophyll Catabolites during Leaf Senescence in Arabidopsis

Nonfluorescent chlorophyll catabolites (NCCs) were described as products of chlorophyll breakdown in Arabidopsis thaliana. NCCs are formyloxobilin-type catabolites derived from chlorophyll by oxygenolytic opening of the chlorin macrocycle. These linear tetrapyrroles are generated from their fluorescent chlorophyll catabolite (FCC) precursors by a nonenzymatic isomerization inside the vacuole of senescing cells. Here, we identified a group of distinct dioxobilin-type chlorophyll catabolites (DCCs) as the major breakdown products in wild-type Arabidopsis, representing more than 90% of the chlorophyll of green leaves. The molecular constitution of the most abundant nonfluorescent DCC (NDCC), At-NDCC-1, was determined. We further identified cytochrome P450 monooxygenase CYP89A9 as being responsible for NDCC accumulation in wild-type Arabidopsis; cyp89a9 mutants that are deficient in CYP89A9 function were devoid of NDCCs but accumulated proportionally higher amounts of NCCs. CYP89A9 localized outside the chloroplasts, implying that FCCs occurring in the cytosol might be its natural substrate. Using recombinant CYP89A9, we confirm FCC specificity and show that fluorescent DCCs are the products of the CYP89A9 reaction. Fluorescent DCCs, formed by this enzyme, isomerize to the respective NDCCs in weakly acidic medium, as found in vacuoles. We conclude that CYP89A9 is involved in the formation of dioxobilin-type catabolites of chlorophyll in Arabidopsis.     

Recognition of the Protein Kinase AVRPPHB SUSCEPTIBLE1 by the Disease Resistance Protein RESISTANCE TO PSEUDOMONAS SYRINGAE5 Is Dependent on S-Acylation and an Exposed Loop in AVRPPHB SUSCEPTIBLE1

The recognition of pathogen effector proteins by plants is typically mediated by intracellular receptors belonging to the nucleotide-binding leucine-rich repeat (NLR) family. NLR proteins often detect pathogen effector proteins indirectly by detecting modification of their targets. How NLR proteins detect such modifications is poorly understood. To address these questions, we have been investigating the Arabidopsis (Arabidopsis thaliana) NLR protein RESISTANCE TO PSEUDOMONAS SYRINGAE5 (RPS5), which detects the Pseudomonas syringae effector protein Avirulence protein Pseudomonas phaseolicolaB (AvrPphB). AvrPphB is a cysteine protease that specifically targets a subfamily of receptor-like cytoplasmic kinases, including the Arabidopsis protein kinase AVRPPHB Susceptible1 (PBS1). RPS5 is activated by the cleavage of PBS1 at the apex of its activation loop. Here, we show that RPS5 activation requires that PBS1 be localized to the plasma membrane and that plasma membrane localization of PBS1 is mediated by amino-terminal S-acylation. We also describe the development of a high-throughput screen for mutations in PBS1 that block RPS5 activation, which uncovered four new pbs1 alleles, two of which blocked cleavage by AvrPphB. Lastly, we show that RPS5 distinguishes among closely related kinases by the amino acid sequence (SEMPH) within an exposed loop in the C-terminal one-third of PBS1. The SEMPH loop is located on the opposite side of PBS1 from the AvrPphB cleavage site, suggesting that RPS5 associates with the SEMPH loop while leaving the AvrPphB cleavage site exposed. These findings provide support for a model of NLR activation in which NLR proteins form a preactivation complex with effector targets and then sense a conformational change in the target induced by effector modification.Pathogen recognition by plants is mediated by both transmembrane cell surface receptors and intracellular receptors (Jones and Dangl, 2006). The latter receptors typically belong to the nucleotide-binding leucine-rich repeat (NLR) superfamily of proteins, which also play a central role in the innate immune systems of many animals, including humans (von Moltke et al., 2013). In plants, most NLR proteins detect pathogen “effector” proteins, which are proteins secreted by pathogens to promote virulence on susceptible hosts. The immune response activated by NLR proteins is thus referred to as effector-triggered immunity. In the majority of examples studied, effector-triggered immunity is accompanied by localized host cell death around the site of pathogen ingress, which is referred to as the hypersensitive response (HR; Goodman and Novacky, 1994).Several NLR proteins have been shown to detect pathogen effector proteins indirectly by detecting the modification of other host proteins mediated by the effectors (DeYoung and Innes, 2006). The best characterized examples of NLR proteins that employ indirect recognition mechanisms are the RESISTANCE TO PSEUDOMONAS MACULICOLA1 (RPM1) and RESISTANCE TO PSEUDOMONAS SYRINGAE2 (RPS2) proteins of Arabidopsis (Arabidopsis thaliana), which detect modification to the RPM1 INTERACTING4 (RIN4) protein (Mackey et al., 2002; Axtell and Staskawicz, 2003), the RESISTANCE TO PSEUDOMONAS SYRINGAE5 (RPS5) protein of Arabidopsis, which detects modification of the AVRPPHB SUSCEPTIBLE1 (PBS1) protein kinase (Ade et al., 2007), and the Pseudomonas resistance and fenthion sensitivity (Prf) protein of tomato (Solanum lycopersicum), which detects modification of the Pseudomonas syringae pv tomato resistance (Pto) protein kinase (Salmeron et al., 1996; Rathjen et al., 1999). Our group has focused on RPS5, which detects the effector protein Avirulence protein Pseudomonas phaseolicolaB (AvrPphB) from Pseudomonas syringae (Simonich and Innes, 1995). AvrPphB functions as a Cys protease (Zhu et al., 2004) and specifically targets a subclass of plant receptor-like cytoplasmic kinases that include PBS1 (Shao et al., 2003; Zhang et al., 2010). AvrPphB likely targets these kinases in order to suppress defense responses induced by cell surface-localized plant immune receptors such as FLAGELLIN SENSITIVE2 (FLS2; Zhang et al., 2010). PBS1 can be coimmunoprecipitated with FLS2, and mutation of PBS1 reduces FLS2-mediated production of hydrogen peroxide and callose deposits (Zhang et al., 2010), confirming that PBS1 functions in defense signaling.Cleavage of PBS1 by AvrPphB is both necessary and sufficient to activate RPS5 (Ade et al., 2007), and null mutations in PBS1 block RPS5 activation (Swiderski and Innes, 2001). Because AvrPphB can cleave multiple closely related kinases in Arabidopsis (Zhang et al., 2010), these observations indicate that RPS5 can distinguish among these kinases, with only PBS1 cleavage activating RPS5. The molecular basis for this specificity is unknown.One contributor to the specificity of RPS5 may be subcellular localization. RPS5 localizes to the plasma membrane (PM), and amino acid substitutions that displace RPS5 from the PM eliminate RPS5-mediated defense responses (Qi et al., 2012). PBS1 is also expected to localize to the PM, because fusion of the N-terminal 100 amino acids of PBS1 to GFP causes GFP to localize to the PM in both Arabidopsis and Nicotiana benthamiana (Takemoto et al., 2012). Consistent with this expectation, PBS1 and RPS5 can be coimmunoprecipitated when transiently overexpressed in N. benthamiana (Ade et al., 2007). Furthermore, AvrPphB is both myristoylated and palmitoylated upon entry into plant cells and localizes to the PM, with PM localization of AvrPphB being required for the activation of RPS5 (Dowen et al., 2009). Although these data all point to a PM localization for PBS1, full-length PBS1 protein has not yet been localized, nor has the functional significance of PBS1 localization been assessed relative to the activation of RPS5.In this study, we demonstrate that PBS1 is targeted to the PM via S-acylation at its N terminus and that PM localization is required for RPS5 activation. We also describe a high-throughput genetic screen for uncovering new mutations in PBS1 that block RPS5 activation, which uncovered four new pbs1 alleles. Lastly, we show that RPS5 distinguishes PBS1 from closely related kinases based on a specific loop in the C-terminal half of PBS1.